Compositions, kits and uses of nucleotides having altered base pairing characteristics

ABSTRACT

The present invention provides improved methods, compositions and kits for amplifying a nucleic acid molecule. Specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with nucleotide residue having altered base-pairing characteristics, such as inosine, hypoxanthine, xanthine, and methylated nucleotide derivatives, so as to more equalize the efficiency with which that oligonucleotide and a second oligonucleotide hybridize to a target molecule, and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in the relative hybridization efficiencies.

This application is a continuation of Ser. No. 09/592,762, filed Jun.13, 2000, abandoned, which is a continuation of Ser. No. 09/227,255,filed Jan. 8, 1999, abandoned, which is a continuation of Ser. No.08/755,736, filed Nov. 25, 1996, U.S. Pat. No. 5,869,251, which is acontinuation of Ser. No. 08/246,921, filed May 20, 1994, U.S. Pat. No.5,578,467, which is a continuation of Ser. No. 07/819,132, filed Jan.10, 1992, abandoned.

FIELD OF THE INVENTION

The present invention is in the field of recombinant DNA technology.This invention is directed to a process for amplifying a nucleic acidmolecule, and to the molecules employed and produced through thisprocess.

BACKGROUND OF THE INVENTION

Assays capable of detecting the presence of a particular nucleic acidmolecule in a sample are of substantial importance in forensics,medicine, epidemiology and public health, and in the prediction anddiagnosis of disease. Such assays can be used, for example, to identifythe causal agent of an infectious disease, to predict the likelihoodthat an individual will suffer from a genetic disease, to determine thepurity of drinking water or milk, or to identify tissue samples. Thedesire to increase the utility and applicability of such assays is oftenfrustrated by assay sensitivity. Hence, it would be highly desirable todevelop more sensitive detection assays.

Nucleic acid detection assays can be predicated on any characteristic ofthe nucleic acid molecule, such as its size, sequence, and, if DNA,susceptibility to digestion by restriction endonucleases, etc. Thesensitivity of such assays may be increased by altering the manner inwhich detection is reported or signaled to the observer. Thus, forexample, assay sensitivity can be increased through the use ofdetectably labeled reagents. A wide variety of such labels have beenused for this purpose. Kourilsky et al. (U.S. Pat. No. 4,581,333)describe the use of enzyme labels to increase sensitivity in a detectionassay. Radioisotopic labels are disclosed by Falkow et al. (U.S. Pat.No. 4,358,535), and by Berninger (U.S. Pat. No. 4,446,237). Fluorescentlabels (Albarella et al., EP 144914), chemical labels (Sheldon III etal., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.4,563,417), modified bases (Miyoshi et al., EP 119448), etc. have alsobeen used in an effort to improve the efficiency with which detectioncan be observed.

Although the use of highly detectable labeled reagents can improve thesensitivity of nucleic acid detection assays, the sensitivity of suchassays remains limited by practical problems which are largely relatedto non-specific reactions which increase the background signal producedin the absence of the nucleic acid the assay is designed to detect.Thus, for some applications, such as for the identification of a pureculture of a bacteria, etc., the concentration of the desired moleculewill typically be amenable to detection, whereas, for other potentialapplications, the anticipated concentration of the desired nucleic acidmolecule will be too low to permit its detection by any of theabove-described assays.

In response to these impediments, a variety of highly sensitive methodsfor DNA amplification have been developed.

One method for overcoming the sensitivity limitation of nucleic acidconcentration is to selectively amplify the nucleic acid molecule whosedetection is desired prior to performing the assay. Recombinant DNAmethodologies capable of amplifying purified nucleic acid fragments havelong been recognized. Typically, such methodologies involve theintroduction of the nucleic acid fragment into a DNA or RNA vector, theclonal amplification of the vector, and the recovery of the amplifiednucleic acid fragment. Examples of such methodologies are provided byCohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, etc.

Other known nucleic acid amplification procedures includetranscription-based amplification systems (Kwoh, D. et al., Proc. Natl.Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras T R et al., PCT appl. WO88/10315 (priority: U.S. patent application Ser. Nos. 064,141 and202,978)). Schemes based on ligation of two (or more) oligonucleotidesin the presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu, D. Y. et al., Genomics 4:560 (1989)).

Miller, H. I. et al., PCT appl. WO 89/06700 (priority: U.S. patentapplication Ser. No. 146,462, filed Jan. 21, 1988), disclose a nucleicacid sequence amplification scheme based on the hybridization of apromoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme was not cyclic; i.e. new templates were not produced from theresultant RNA transcripts.

Davey, C. et al. (European Patent Application Publication no. 329,822)disclose a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′-to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Methods that include a transcription step, e.g. that of Davey, C. et al.(European Patent Application Publication no. 329,822), can increaseproduct by more than a factor of 2 at each cycle. Indeed, as 100 or moretranscripts can be made from a single template, factors of increase of100 or more are theoretically readily attainable. Furthermore, if allsteps are performed under identical conditions, no molecule which hasfinished a particular step need “wait” before proceeding to the nextstep. Thus amplifications that are based on transcription and that donot require thermo-cycling are potentially much faster thanthermo-cycling amplifications which are based on template-dependentprimer extension.

In methods which amplify a nucleic acid molecule by template dependentextension, the nucleic acid molecule is used as a template for extensionof a nucleic acid primer in a reaction catalyzed by polymerase. Forexample, Panet and Khorana (J. Biol. Chem. 249:5213-5221 (1974) whichreference is incorporated herein by reference) demonstrated thereplication of deoxyribopoly-nucleotide templates bound to cellulose.Kleppe et al. (J. Mol. Biol. 56:341-361 (1971) which reference isincorporated herein by reference) disclosed the use of double andsingle-stranded DNA molecules as templates for the synthesis ofcomplementary DNA.

The most widely used method of nucleic acid amplification, the“polymerase chain reaction” (“PCR”), involves template dependentextension (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017,EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No.4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al.,U.S. Pat. No. 4,683,194), which references are incorporated herein byreference). PCR achieves the amplification of a specific nucleic acidsequence using two oligonucleotide primers complementary to regions ofthe sequence to be amplified. Extension products incorporating theprimers then become templates for subsequent replication steps.

The polymerase chain reaction provides a method for selectivelyincreasing the concentration of a nucleic acid molecule having aparticular sequence even when that molecule has not been previouslypurified and is present only in a single copy in a particular sample.The method can be used to amplify either single or double stranded DNA.The essence of the method involves the use of two oligonucleotides toserve as primers for the template-dependent, polymerase mediatedreplication of the desired nucleic acid molecule.

The precise nature of the two oligonucleotide primers of the PCR methodis critical to the success of the method. As is well known, a moleculeof DNA or RNA possesses directionality, which is conferred through the5′→3′ linkage of the sugar-phosphate backbone of the molecule. Two DNAor RNA molecules may be linked together through the formation of aphosphodiester bond between the terminal 5′ phosphate group of onemolecule and the terminal 3′ hydroxyl group of the second molecule.Polymerase dependent amplification of a nucleic acid molecule proceedsby the addition of a 5′ nucleoside triphosphate to the 3′ hydroxyl endof a nucleic acid molecule. Thus, the action of a polymerase extends the3′ end of a nucleic acid molecule. These inherent properties areexploited in the selection of the two oligonucleotide primers of thePCR. The oligonucleotide sequences of the two primers of the PCR methodare selected such that they contain sequences identical to, orcomplementary to, sequences which flank the sequence of the particularnucleic acid molecule whose amplification is desired. More specifically,the nucleotide sequence of the “first” primer is selected such that itis capable of hybridizing to an oligonucleotide sequence located 3′ tothe sequence of the desired nucleic acid molecule, whereas thenucleotide sequence of the “second” primer is selected such that itcontains a nucleotide sequence identical to one present 5′ to thesequence of the desired nucleic acid molecule. Both primers possess the3′ hydroxyl groups which are necessary for enzyme mediated nucleic acidsynthesis.

In the polymerase chain reaction, the reaction conditions are cycledbetween those conducive to hybridization and nucleic acidpolymerization, and those which result in the denaturation of duplexmolecules. In the first step of the reaction, the nucleic acids of thesample are transiently heated, and then cooled, in order to denature anydouble stranded molecules which may be present. The “first” and “second”primers are then added to the sample at a concentration which greatlyexceeds that of the desired nucleic acid molecule. When the sample isincubated under conditions conducive to hybridization andpolymerization, the “first” primer will hybridize to the nucleic acidmolecule of the sample at a position 3′ to the sequence of the desiredmolecule to be amplified. If the nucleic acid molecule of the sample wasinitially double stranded, the “second” primer will hybridize to thecomplementary strand of the nucleic acid molecule at a position 3′ tothe sequence of the desired-molecule which is the complement of thesequence whose amplification is desired. Upon addition of a polymerase,the 3′ ends of the “first” and (if the nucleic acid molecule was doublestranded) “second” primers will be extended. The extension of the“first” primer will result in the synthesis of a DNA molecule having theexact sequence of the complement of the desired nucleic acid. Extensionof the “second” primer will result in the synthesis of a DNA moleculehaving the exact sequence of the desired nucleic acid.

The PCR reaction is capable of exponential amplification of specificnucleic acid sequences because the extension product of the “first”primer contains a sequence which is complementary to a sequence of the“second” primer, and thus will serve as a template for the production ofan extension product of the “second” primer. Similarly, the extensionproduct of the “second” primer, of necessity, contain a sequence whichis complementary to a sequence of the “first” primer, and thus willserve as a template for the production of an extension product of the“first” primer. Thus, by permitting cycles of hybridization,polymerization, and denaturation, a geometric increase in theconcentration of the desired nucleic acid molecule can be achieved.Reviews of the polymerase chain reaction are provided by Mullis, K. B.(Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K.,et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al.(Met. Enzymol. 155:335-350 (1987), which references are incorporatedherein by reference).

PCR technology is useful in that it can achieve the rapid and extensiveamplification of a polynucleotide molecule. However, the method requiresthe preparation of two different primers which hybridize to twooligonucleotide sequences flanking the target sequence. Theconcentration of the two primers can be rate limiting for the reaction.Although it is not essential that the concentration of the two primersbe identical, a disparity between the concentrations of the two primerscan greatly reduce the overall yield of the reaction.

All of the above amplification procedures depend on the principle thatan end product of a cycle is functionally identical to a startingmaterial. Thus, by repeating cycles, the nucleic acid is amplifiedexponentially.

Methods that use thermo-cycling, e.g. PCR or Wu, D. Y. et al. (Genomics4:560 (1989)), have a theoretical maximum increase of product of 2-foldper cycle, because in each cycle a single product is made from eachtemplate. In practice, the increase is always lower than 2-fold. Furtherslowing the amplification is the time spent in changing the temperature.Also adding delay is the need to allow enough time in a cycle for allmolecules to have finished a step. Molecules that finish a step quicklymust “wait” for their slower counterparts to finish before proceeding tothe next step in the cycle; to shorten the cycle time would lead toskipping of one cycle by the “slower” molecules, leading to a lowerexponent of amplification.

One disadvantage of PCR is that it requires the use of two primers, andthus requires that sequence information be available for two regions ofthe target molecule. This is often a significant constraint. In somesituations, only the amino acid sequence encoded by a target sequence isknown. To amplify the target sequence, it is necessary to employ sets ofdegenerate primers (corresponding to each of the possible sequencescapable of encoding the amino acid sequence coded for by the two regionsof the target molecule). The use of such degenerate primer sets cancause significant priming errors, and thus an decrease amplificationefficiency. One means of decreasing the number of members in the primersets when conducting PCR amplification is through the use of primerscontaining deoxyinosine at positions of ambiguity (Patil, R. V., Nucl.Acids Res. 18:3080(1990); Fordham-Skelton, A. P., et al., Molec. Gen.Genet. 221:134-138 (1990); both of which references are hereinincorporated by reference).

A second significant disadvantage of the PCR reaction is that when twodifferent primers are used, the reaction conditions chosen must beselected such that both primers “prime” with similar efficiency. Sincethe two primers necessarily have different sequences, this requirementcan constrain the choice of primers and require considerableexperimentation. Furthermore, if one tries to amplify two differentsequences simultaneously using PCR (i.e. using two sets of two primers),the reaction conditions must be optimized for four different primers.

SUMMARY OF THE INVENTION

The present invention provides an improved method for equalizing thehybridization efficiency of the primers used in a PCR reaction. It thuscomprises an improvement in PCR amplification. The invention achievesthis goal by employing a primer molecule which contains pre-determinednucleotides having altered base pairing characteristics.

In detail, the invention provides a method for amplifying theconcentration of a nucleic acid molecule using two primers, comprisingthe steps:

(a) performing the template-dependent extension of a first primer, theprimer being hybridized to a first strand of the molecule, wherein theextension forms a second strand of a nucleic acid molecule complementaryto the first strand;

(b) performing the template-dependent extension of the second strand, byextending a second primer, the primer being hybridized to the secondstrand of the molecule, wherein the extension forms a copy of the firststrand of the nucleic acid molecule;

(c) performing the template-dependent extension of the copy of the firststrand, to thereby form a copy of the second strand of the nucleic acidmolecule;

(d) repeating steps (a), (b), and (c), to thereby achieve theamplification of the nucleic acid molecule;

wherein at least one of the first and second primers contains at leastone deoxyinosine residue, and wherein the first and second primers haveequivalent efficiency of primer extension.

The invention also provides the embodiments of the above method whereinthe nucleic acid molecule is an RNA or a DNA molecule, and wherein suchmolecule is either single-stranded or double-stranded.

The invention also provides the embodiments of the above methods whereinonly one of the primers contains at least one deoxyinosine residue, andwherein both of the primers contain at least one deoxyinosine residue.

The invention also provides the embodiment of the above methods whereinthe nucleic acid molecule being amplified is polyadenylated at its 3′end, and wherein one of the primers contains a poly-T sequence, and theother of the primers contains at least one deoxyinosine residue.

The invention also provides the embodiment of the above methods whereinthe nucleic acid molecule being amplified, copy thereof or complementarycopy thereof has been extended to contain a 3′ sequence, and wherein oneof the primers is capable of hybridizing to the 3′ sequence, the primercontaining at least one deoxyinosine residue.

The invention also provides the embodiment of the above methods whereinat least one of the primers is extended using a thermostable DNApolymerase, such as Taq polymerase.

The invention also provides a kit for amplifying a nucleic acid moleculecontaining:

a first container containing a primer, the primer being capable ofhybridizing to the nucleic acid molecule, and containing at least onedeoxyadenosine residue; and

a second container containing an enzyme capable of adding a C nucleotideto the nucleic acid molecule, the C nucleotide being capable of basepairing with the deoxyinosine residue of the primer.

The invention also provides for the above kit which additionallycontains a third container containing a thermostable DNA polymerase,such as Taq polymerase capable of extending the primer of the firstcontainer, when the primer is hybridized to a sequence containing the Cresidue added by the enzyme of the second container.

The invention also provides a kit for amplifying a nucleic acid moleculecontaining:

a first container containing a first primer, the primer being capable ofhybridizing to the nucleic acid molecule, and containing at least onedeoxyinosine residue; and

a second container containing a second primer; whereintemplate-dependent extension of the first primer produces a secondnucleic acid molecule which is capable of hybridizing to the secondprimer, and wherein template-dependent extension of the second primerproduces a copy of the first nucleic acid molecule.

The invention also provides the above kit which additionally contains athird container containing a thermostable DNA polymerase, such as Taqpolymerase, capable of extending either the primer of the firstcontainer, or the primer of the second container when the primer ishybridized to a nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show a depiction of the 3′ RACE reaction.

FIGS. 2A-2E show a depiction of the 5′ RACE reaction.

FIGS. 3A-3E show the use of inosine in the 3′ RACE reaction.

FIGS. 4A-4E show the use of inosine in the 5′ RACE reaction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved method for amplifying adesired nucleic acid molecule in a sample. Such samples may includebiological samples derived from a human or other animal source (such as,for example, blood, stool, sputum, mucus, serum, urine, saliva,teardrop, a biopsy sample, an histology tissue sample, a PAP smear, amole, a wart, an agricultural product, waste water, drinking water,milk, processed foodstuff, air, etc.) including samples derived from abacterial or viral preparation, as well as other samples (such as, forexample, agricultural products, waste or drinking water, milk or otherprocessed foodstuff, air, etc.).

The method, provided by the present invention, for amplifying a desirednucleic acid molecule in a sample, may be used to amplify any desirednucleic acid molecule. Such molecules may be either DNA or RNA. Themolecule may be in either a double-stranded or single-stranded form.However, if the nucleic acid is double-stranded at the start of theamplification reaction it is preferably first treated to render the twostrands into a single-stranded, or partially single-stranded, form.Methods are known to render double-stranded nucleic acids intosingle-stranded, or partially single-stranded, forms, such as heating,or by alkali treatment, or by enzymatic methods (such a by helicaseaction, etc.), or by binding proteins, etc. General methods foraccomplishing this treatment are provided by Maniatis, T., et al. (In:Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratories,. Cold Spring Harbor, N.Y. (1982)), and by Haymes, B. D.,et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985)), which references are herein incorporated byreference.

Macromolecular entities that contain nucleic acid other thandouble-stranded DNA, or single-stranded DNA, such as single-strandedRNA, double-stranded RNA or mRNA are capable of being amplified by themethod of the invention. For example, the RNA genomes of certain virusescan be converted to DNA by reaction with enzymes such as reversetranscriptase (Maniatis, T. et al., Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory, 1982; Noonan, K. F. et al.,Nucleic Acids Res. 16:10366 (1988)). The product of the reversetranscriptase reaction may then be amplified according to the invention.

The nucleic acid molecules which may be amplified in accordance with thepresent invention may be homologous to other nucleic acid moleculespresent in the sample (for example, it may be a fragment of a humanchromosome isolated from a human cell biopsy, etc.). Alternatively, themolecule may be heterologous to other nucleic acid molecules present inthe sample (for example, it may be a viral, bacterial, or fungal nucleicacid molecule isolated from a sample of human blood, stools, etc.). Themethods of the invention are capable of simultaneously amplifying bothheterologous and homologous molecules. For example, amplification of ahuman tissue sample infected with a virus may result in amplification ofboth viral and human sequences.

The present methods do not require that the molecules to be amplifiedhave any particular sequence or length. In particular, the moleculeswhich may be amplified include any naturally occurring procaryotic (forexample, pathogenic or non-pathogenic bacteria, Escherichia, Salmonella,Clostridium, Agrobacter, Staphylococcus and Streptomyces, Streptococcus,Rickettsiae, Chlamydia, Mycoplasma, etc.), eukaryotic (for example,protozoans and parasites, fungi, yeast, higher plants, lower and higheranimals, including mammals and humans) or viral (for example, Herpesviruses, HIV, influenza virus, Epstein-Barr virus, hepatitis virus,polio virus, etc.) or viroid nucleic acid. The nucleic acid molecule canalso be any nucleic acid molecule which has been or can be chemicallysynthesized. Thus, the nucleic acid sequence may or may not be found innature.

“Primer” as used herein refers to a single-stranded oligonucleotide or asingle-stranded polynucleotide that is extended by covalent addition ofnucleotide monomers during amplification. Nucleic acid amplificationoften is based on nucleic acid synthesis by a nucleic acid polymerase.Many such polymerases require the presence of a primer that can beextended to initiate such nucleic acid synthesis. A primer is typically11 bases or longer; most prefererably, a primer is 17 bases or longer.

“Reaction” denotes a liquid suitable for conducting a desired reaction(such as amplification, hybridization, cDNA synthesis, etc.).

“Amplification” as used herein refers to an increase in the amount ofthe desired nucleic acid molecule present in a sample. “Substantialamplification” refers to greater than about three-fold amplification.Any of the primer-extension amplification methods discussed above may beimproved in accordance with the present invention.

As used herein, two sequences are said to be able to hybridize or annealto one another if they are capable of forming an anti-paralleldouble-stranded nucleic acid structure. Conditions of nucleic acidhybridization suitable for forming such double stranded structures aredescribed by Maniatis, T. et al. (In: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982)), and by Haymes, B. D., et al. (In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985), both hereinincorporated by reference). Two sequence are said to be “complementary”to one another if they are capable of hybridizing to one another to forma stable anti-parallel double-stranded nucleic acid structure. Thus, thesequences need not exhibit precise complementarity, but need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure. Thus, departures from completecomplementarity are permissible, so long as such departures are notsufficient to completely preclude hybridization to form adouble-stranded structure. Hybridization of a primer to a complementarystrand of nucleic acid is a prerequisite for its template-dependentpolymerization with polymerases. Factors (see Maniatis, T., et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1982), and Haymes, B. D., et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985))which affect the base pairing of primers to their complementary nucleicacids subsequently affect priming efficiency (i.e. the relative rate ofthe initiation of priming by the primer). The nucleotide composition ofa primer can affect the temperature at which annealing is optimal andtherefore can affect its priming efficiency.

The methods of the present invention permit one to adjust thehybridization efficiency of the primers used to amplify a nucleic acidmolecule. Such adjustment may either increase or decrease the differencebetween the respective hybridization efficiencies of two primers. Themethods of the present invention thus permit one to equalize therespective hybridization efficiencies of the two primers. Severalfactors must be considered in order to determine the efficiency ofhybridization between a primer and a target molecule. At the simplestlevel, the efficiency is determined by the length of the primer, theconcentration of the primer, the temperature and the ionic strength ofthe reaction. It is also influenced by the sequence complexity of theprimer, and specifically, by the number of hydrogen bonds which willform between the primer and the template. As stated above, the basepairing of an A to a T will form two hydrogen bonds; the base pairing ofa G to a C will form three hydrogen bonds. Thus, at a firstapproximation, it is possible to more closely hybridize two differentprimers to two regions of a target molecule by adjusting the length andsequence complexity of the primers so that they contain the same numberof hydrogen bonds. Unfortunately, additional factors, such as secondarystructure, stacking energy, cooperativity in binding, etc., complicatethe analysis. Thus, a determination of the conditions needed to ensurethat two primers hybridize with equal efficiency requires a multi-factoranalysis. Methods for determining relative primer efficiency aredisclosed by Breslauer, K. J. et al. (Proc. Natl. Acad. Sci. (U.S.A.)83:3746-3750 (1986)), Freier, S. M. et al. (Proc. Natl. Acad. Sci.(U.S.A.) 83:9373-9377 (1986)), Rychlik, W. et al. (Nucl. Acids Res.17:8543-8551 (1989)), Lathe, R. (J. Molec. Biol. 183:1-12 (1986)),Sambrook, J. et al. (Molecular Cloning (A Laboratory Manual), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), vol. 2, pp.11-18), Schildkraut, C. et al. (Biopolymers 3:195-208 (1965)), Baldino,F. et al. (Meth. Enzymol. 168:761-777 (1989)), and in the Handbook ofBiochemistry and Molecular Biology (Fasman, G. D., Ed.), Third Edition(1975), Nucleic Acids, Vol. 1, pp. 589, CRC Press, Cleveland, Ohio; allof which references are herein incorporated by reference. Mostpreferably, a determination of relative primer efficiency is performedusing a computer program, such as “Oligo™ Primer Analysis Software(National Biosciences, Inc., Hamel, Minn.).

The improvement provided by the present invention results from using“pre-determined” nucleotides having altered base pairing characteristicsin at least one of the primer molecules to equalize the efficiency ofhybridization between (1) that primer molecule and its complementsequence on the target molecule, and (2) a second primer molecule andits complement sequence on the target molecule. The primers of thepresent invention are preferably 15-50 residues in length, althoughshorter or longer primer sequences can be employed.

DNA typically contains a polynucleotide composed of the 4 “natural”bases: A (adenine), T (thymine), C (cytosine), and G (guanine). Thehydrogen bonding (or base pairing) among these nucleotides creates thedouble-stranded structure of a DNA molecule. An A-containing residuebase pairs to a T-containing residue through the formation of twohydrogen bonds; a G-containing residue base pairs to a C-containingresidue through the formation of three hydrogen bonds.

The term “pre-determined nucleotides having altered base pairingcharacteristics” is intended to refer to nucleotides which have basesother than the A, T, C or G naturally found in DNA. Although thepre-determined nucleotides will be capable of hydrogen bonding withnaturally occurring nucleotides (such as the A, T, C or G-containingnucleotides of the template), it will form fewer hydrogen bonds withsuch nucleotides than would other naturally occurring nucleotides.

A nucleotide containing deoxyinosine (“dI”) is a preferred example of asuch a pre-determined nucleotide containing the base, inosine. It iscapable of forming two hydrogen bonds with either A, C, T, or G (Barker,R., Organic Chemistry of Biological Molecules, Prentice-Hall, N.J.(1971)). Thus, in a preferred embodiment, when I is used in a primer (ortemplate) in lieu of G or, in lieu of C, the base pairing efficiency isaltered.

Other examples of “pre-determined” nucleotides are those which containhypoxanthine or xanthine (each useful, for example, in lieu of G, toform two hydrogen bonds when base pairing with C), or those whichcontain methylated derivatives of naturally occurring bases (forexample, 7-methylguanine, etc.).

In accordance with the methods of the present invention, nucleic acidamplification, such as through two primer mediated PCR, is achievedusing at least one primer containing at least one of the above-described“pre-determined” nucleotides. The position, type and number of“pre-determined” nucleotide(s) in the primer sequence containing the“pre-determined” nucleotide are selected such that the efficiency ofprimer extension of that primer is equivalent to the efficiency ofprimer extension of the second primer.

As used herein, the term “equivalent efficiency of primer extension” isintended to refer to the ability of one primer, relative to a secondprimer, to hybridize to a complementary sequence on a template molecule,and to serve as a substrate for template-dependent primer extension by aDNA or RNA polymerase. Primer extension is said to be “templatedependent” when the sequence of the newly synthesized strand of nucleicacid is dictated by complementary base pairing. A “polymerase” is anenzyme that is capable of incorporating nucleoside triphosphates toextend a 3′ hydroxyl group of a nucleic acid molecule, if that moleculehas hybridized to a suitable template nucleic acid molecule. Polymeraseenzymes are discussed in Watson, J. D., In: Molecular Biology of theGene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), whichreference is incorporated herein by reference, and similar texts.Examples of polymerases include the large “Klenow” fragment of E. coliDNA polymerase I; Taq polymerase (Cetus); bacteriophage T7 DNA or RNApolymerase, etc. A preferred DNA polymerase is Taq polymerase (Cetus).

When an enzymatic reaction, such as a polymerization reaction, is beingconducted, it is preferable to provide the components required for suchreaction in “excess” in the reaction vessel. “Excess” in reference tocomponents of the amplification reaction refers to an amount of eachcomponent such that the ability to achieve the desired amplification isnot substantially limited by the concentration of that component.

Conditions or agents which increase the rate or the extent of priming,primer elongation, or strand displacement, may increase the extent ofthe amplification obtained with the methods of the present invention.For instance, the addition of helicases or single-stranded nucleic acidbinding proteins may increase the strand displacement rate of a DNApolymerase, or may allow the use of a DNA polymerase that might notordinarily give substantial amplification.

It is desirable to provide to the assay mixture an amount of requiredcofactors such as Mg⁺⁺, and dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTPor and the “pre-determined” nucleoside triphosphates in sufficientquantity to support the degree of amplification desired.

Extension of the primer in, for example, a cDNA-containing reaction, maybe done with the same reverse transcriptase used to make cDNA.Alternatively, one can add a new DNA polymerase for cDNA extension.Removal of the RNA from the cDNA is preferably done by an RNase Htreatment, or by the action of a helicase, but can be done by physicaldenaturation, e.g. heat, formamide, or alkali (high pH). In the lattercase, if kinetics of renaturation are sufficiently high, this step mustbe followed by physical separation of the cDNA and RNA or by degradationof the RNA, e.g. by RNase or alkali. Note that sufficiently harsh alkalitreatment may deaminate dC to form dU, causing a mutation.

Reverse transcription can be done with a reverse transcriptase that hasRNase H activity. If one uses an enzyme having RNase H activity, it maybe possible to omit a separate RNase H digestion step, by carefullychoosing the reaction conditions.

All of the enzymes used in this amplification reaction may be activeunder the same reaction conditions. Indeed, buffers exist in which allenzymes are near their optimal reaction conditions. Therefore, theamplification process of the present invention can be done in a singlereaction volume without any change of conditions such as addition ofreactants or temperature cycling. Thus, though this process has severalsteps at a molecular level, operationally it may have a single step.Once the reactants are mixed together, one need not add anything orchange conditions, e.g. temperature, until the amplification reactionhas exhausted one or more components. During this time, the nucleic acidsequence being amplified will have been increased many-fold. The levelof increase will be sufficient for many purposes; however, for somepurposes the reaction may have to be repeated with fresh components toachieve the desired level of amplification.

As discussed above, the degree of amplification obtained through the useof PCR is limited if the two primers do not have equivalent efficiencyof primer extension. Such a situation is frequently encountered,especially in amplification protocols such as RACE, anchored PCR,one-sided PCR, etc. (Frohman, M. A. et al., Proc. Natl. Acad. Sci.(U.S.A.) 85:8998-9002 (1988); Ohara, O. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86:5673-5677 (1989), both of which references are hereinincorporated by reference). In brief, these procedures facilitate therecovery of full-length cDNAs from rare transcripts. The RACE procedureresults in the amplification of sequences 3′ and 5′ of a particularsequence known to be present in a desired molecule.

For example in the amplification of mRNA or cDNA molecule having a 3′poly-A region, two primers are typically employed. The first primercontains poly-T, and the second primer contains a sequence complementaryto an internal gene sequence of the mRNA or cDNA molecule. Thisprocedure is referred to as a 3′ RACE (FIG. 1). As shown in FIG. 1A,hybridization with the first primer 3′ poly T 5′ is capable ofhybridizing to the poly-A sequence (FIG. 1B). After primer extension andstrand separation, the structure shown in FIG. 1C is obtained. AfterHybridization with the first primer and with a second primer capable ofhybridizing to a known sequence, the structure shown in FIG. 1D isobtained. Primer extension of both primers yields the structure shown inFIG. 1E.

Similarly, it is often desirable to amplify a target molecule for whichonly one sequence specific primer is available. This can be accomplishedby adding a nucleotide sequence to one end of the target molecule, orcomplementary copy thereof, and then using a primer which iscomplementary to the added nucleotide sequence (FIG. 2). The targetmolecule (FIG. 2A) is hybridized with a first primer capable ofhybridizing to a known sequence (FIG. 2B). After primer extension, andstrand separation or RNAse R degradation of target template thestructure shown in FIG. 2C is obtained. This structure is treated withterminal deoxynucleotidyl transferase and dC to add a poly-dC tail tothe extension product, and the other strand is of no further interest,or has been destroyed by the RNAse H treatment (FIG. 2D). The poly-dctailed product is amplified by PCR using a poly-dG primer, and a primercapable of hybridizing to the region of known sequence (FIG. 2E). Inpractice, any nucleotide (A, C, T, or G) could have been used to producethe homopolymer tail and be amplified using a complementaryoligonucleotide primer.

In either of the above examples, the two primers will not have the sameefficiency of primer extension. In the first example, the primer havinga poly-T sequence will have a lower Tm than the second primer. In thesecond example, the poly-dG primer will have a higher Tm than the otherprimer.

It should be noted that RACE procedures may generate artifact productsunless nested PCR is done. Nested PCR is disclosed in U.S. Pat. Nos.4,683,195 and 4,683,202, herein incorporated by reference. Nested PCRmay also be used to eliminate non-specific amplification products. Notethat nested PCR often refers to PCR with primers “nested” at both endsof the sequence, i.e. PCR conducted using 4 oligonucleotides.

As will be recognized, in both of the preceding examples a homopolymerprimer (i.e. poly-T or poly-dG) is used in conjunction with a secondprimer having a greater sequence complexity. The present inventionpermits one to make the annealing efficiencies of the two primersequivalent by replacing some or all of those natural residues of theprimers which form 3 hydrogen bonds with pre-determined residues capableof forming only 2 hydrogen bonds. The number, type and position of thepre-determined substitute residues is determined (as described above)such that the two primers used in the amplification have equivalentannealing efficiency.

The primer molecules themselves can be extended by the terminaldeoxynucleotidyl transferase. This is generally an undesired reaction,since it leads to the formation of “primer-dimers” and decreases theefficiency of target molecule amplification. Thus, it is generallypreferable to remove any primer molecules from the reaction prior to thepolynucleotide kinase extension step. Once the step has been completed,the primers may be returned to the reaction. It is thereafterunnecessary to remove the primers after subsequent steps of theamplification.

The use of such replacement residues in the amplification of apolyadenylated cDNA or mRNA is illustrated in FIG. 3. The figure isidentical to FIG. 1 except for the presence of inosine (represented as a“*”) in the primer and extension product.

The use of the “pre-determined” nucleotides in the second primer lowersthe Tm of that primer, thus permitting it to be equivalent to the Tm ofthe poly-T primer.

The use of “pre-determined” replacement residues in the amplification ofa non-polyadenylated cDNA or mRNA sequence is illustrated below (withthe designation “GG*GG*G” referring to a primer having at least one“pre-determined” nucleotide such as deoxyinosine) FIG. 4). The figure isidentical to FIG. 2 except for the presence of inosine (represented as a“*”) in the primer and extension product.

The use of the “pre-determined” nucleotides in the second primer lowersthe Tm of that primer, thus permitting it to be equivalent to the Tm ofthe first primer. Note that all of the amplified sequences between thetwo primer sequences will be the initially present, desired sequence.

The present invention is also applicable to amplification proceduresother than PCR (such as, for example, “Ligation Chain Reaction” (“LCR”)described by, for example, Wu, D. Y. et al., Genomics 4:560 (1989), orthe methods of Miller, H. I. (WO 89/06700), Davey, C. et al. (EP329,822), Kwoh, D. (Proc. Natl. Acad. Sci. (USA) 86:1173 (1989)), etc.).Other suitable methods for amplifying nucleic acid based on ligation oftwo oligonucleotides after annealing to complementary nucleic acids areknown in the art.

The method of this invention can be used to adjust (for example, toequalize) the annealing of the oligonucleotides prior to ligation. Theligase based methods have been used for discrimination of targetmolecules which are different by a single nucleotide. The methods of thepresent invention are also applicable for adjusting or equalizing theannealing of oligonucleotides.

The present invention thus provides a method for adjusting thehybridization efficiency of an oligonucleotide (preferably a primer) ofpredetermined sequence complementary to a target nucleic acid molecule(most preferably a cDNA molecule), which comprises: A) employing as theoligonucleotide an oligonucleotide wherein at least one residue is adeoxyinosine residue; and B) permitting the oligonucleotide to hybridizewith the target molecule. In this method, the presence of thedeoxyinosine residue effects the adjustment of the hybridizationefficiency. As will be appreciated, this adjustment can either increaseor decrease the hybridization efficiency of the respective molecules.

In the above-described ligation chain reaction (LCR) method, a mutationin a target molecule can be detected by using two oligonucleotides eachcapable of hybridizing to adjacent positions on the target molecule,such that the positions flank the site of potential mutation. Thesequences of the oligonucleotides is such that if the mutation ispresent, the hybridized molecules will be able to anneal to one another.As will be appreciated, it is readily possible to employ oligonucleotidesequences such that the hybridized molecules will be able to anneal toone another if the mutation is not present. Thus, the capacity of theoligonucleotides to ligate to one another is “probative” of the presenceof the mutation.

The present invention permits one to adjust the relative hybridizationefficiency of the two oligonucleotides, by incorporating deoxyinosineinto one or both oligonucleotides. It is preferable to adjust thisrelative efficiency to equalize the respective efficiencies of the twooligonucleotides. Such equalization can compensate for operationalconstraints caused by differences in the respective G-C content, size,concentration, etc. between the two oligonucleotides.

In a preferred embodiment, the ligation of the oligonucleotides resultsin the formation of a primer molecule that can be enzymatically extendedto form a complement of the target molecule. Such amplification may beby or include PCR, but will most preferably be mediated by an isothermalextension of the primer to produce the complement to the targetmolecule.

The present invention may be combined with many other processes in thearts of molecular biology to achieve a specific end. Of particularinterest is purifying the target sequence from the other sequences inthe sample. This can be accomplished most advantageously by annealingthe nucleic acid sample to an oligonucleotide that is complementary tothe target and is immobilized on a solid support. A convenient supportwould be a micro-bead, especially a magnetic micro-bead. After being sobound, the non-target sequences could be washed away, resulting in acomplete or a partial purification.

After an amplification is performed, one may wish to detect anyamplification products produced. Any number of techniques known to theart may be adapted to this end without undue experimentation.Particularly advantageous in some situations is the capture ofamplification products by an oligonucleotide complementary to a sequencedetermined by the target sequence, the oligonucleotide being bound to asolid support such as a magnetic micro-bead. Preferably, thisoligonucleotide's sequence does not overlap with that of anyoligonucleotide used to purify the target before the amplification.RNA:DNA hybrids formed may then be detected by antibodies that bindRNA:DNA heteroduplexes. Detection of the binding of such antibodies canbe done by a number of methods well known to the art.

Alternatively, amplified nucleic acid can be detected by gelelectrophoresis, hybridization, or a combination of the two, as is wellunderstood in the art. Those in the art will find that the presentinvention can be adapted to incorporate many detection schemes.

Sequences amplified according to the methods of the invention may bepurified (for example, by gel electrophoresis, by column chromatography,by affinity chromatography, by hybridization, etc.) and the fractionscontaining the purified products may be subjected to furtheramplification in accordance with the methods of the invention.

The present invention includes articles of manufacture, such as “kits.”Such kits will, typically, be specially adapted to contain in closecompartmentalization a first container which contains a pre-determinednucleotide or a primer containing a pre-determined nucleotide (such asdI); a second container which contains an enzyme capable of adding anucleotide (capable of base pairing with the pre-determined nucleotide)to a target nucleic acid molecule. The kit may additionally containbuffers, polymerase or other enzymes, instructional brochures, and thelike.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1

Methods describing the application of the polymerase chain reaction tothe amplification of cDNA-ends derived from low copy number mRNAs usinga single gene specific primer have been described. Reported as “5′-RACE”(Frohman, M. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:8998 (1988)),“anchor PCR” (Loh, E. Y., et al., Science 243:217 (1989)), and“one-sided PCR” (Ohara, O. et al., Proc. Natl. Acad. Sci. (U.S.A.)86:5673 (1989)), these methods, which facilitate the capture of sequencefrom 5′-ends of mRNA proceed through the following steps: (1) conversionof specific cDNA using a gene-specific oligonucleotide primer (GSP1);(2) homopolymeric tailing of cDNA with terminal deoxynucleotidyltransferase (TdT); and (3) PCR amplification of tailed cDNA using an“anchor primer” specific for the homopolymer tail, and a second “nested”gene-specific oligonucleotide (“GSP2”) which primes upstream of theoriginal primer used for cDNA synthesis.

The efficacy of deoxyinosine-containing oligonucleotides to primedC-tailed cDNA in 5′-RACE procedures was tested using a model systememploying an in vitro transcribed RNA analyte added to total RNAisolated from HeLa cells. The enhanced ability of adeoxyinosine-containing anchor primer to carry out specific PCRamplification of 5′-ends of low copy mRNA from complex mixtures wasdemonstrated in this system by direct comparison to amplification withan oligo-dG anchor primer.

Materials and Methods

General items. SUPERSCRIPT™ RNase H⁻ reverse transcriptase (RT), E. coliRNase H and TdT were from Life Technologies, Inc. Taq DNA polymerase waspurchased from Perkin-Elmer Cetus. Buffer components and generalreagents were from either GIBCO BRL or Sigma. Deoxyribonucleosidetriphosphates and ribonucleoside triphosphates were purchased as 100 mMsolutions from Pharmacia.

Oligonucleotides. Oligonucleotides were synthesized by phosphoramiditechemistry using an Applied Biosystems model 380A synthesizer.Oligonucleotides greater than 35 bases were purified by denaturingpolyacrylamide gel electrophoresis and were eluted from the gel matrixessentially as described by Smith, H. O. (Methods Enzymol. 65:371(1980)). Other oligonucleotides were used as machine grade preparationsafter removal of salts and organics by PD-10 chromatography. Molarextinction coefficients and T_(m) calculations for each oligonucleotidewere calculated using the OLIGO computer program from NationalBiosciences (Rychlik, W. and Rhodes, R. E., Nucleic Acids Res. 17:8543(1989)). For the GSP1 primer, a 24-mer oligonucleotide was employed. TheGSP2 primer was 21 nucleotides long. Two anchor primers (a “G-AnchorPrimer” and a GI-Anchor Primer”) were employed. Both molecules had anidentical 18 residue long sequence that was complementary to the target,and located immediately 5′ to a poly-G sequence of 15 residues(“G-Anchor Primer”) or a poly-GI sequence of 16 residues (“GI-AnchorPrimer”). The poly-G and poly-GI sequences differed in that nucleotides4, 5, 9, 10, 14 and 15 of the poly-GI sequence of the GI-Anchor Primerwere dI, whereas in the G-Anchor Primer, all of the nucleotides of thepoly-G sequence were dG.

Preparation of RNA Analyte. RNA analyte for 5′-RACE was an in vitrotranscription product from the gene for chloramphenicol acetyltransferase (CAT) (Horinouchi, S. and Weisblum, B., J. Bacteriol.150:815 (1982)). In vitro transcription was performed using T7 RNApolymerase (GIBCO BRL) according to the manufacturer's recommendations.DNA template was degraded using RNase-free DNase (GIBCO BRL). RNA wasextracted once using a 50:50 mixture of phenol:chloroform and purifiedby Sephadex G-50 (Pharmacia) chromatography. CAT RNA was diluted in DEPCtreated water, and stored at −70° C.

Preparation of Total HeLa RNA. Total RNA was isolated from HeLa cells byguanidinium thiocyanate extraction and equilibrium centrifugation inCsTFA essentially as described by Okayama, H. et al. (Methods Enzymol.154:3 (1987)) using an RNA Extraction Kit from Pharmacia.

5′-RACE. Varying amounts of in vitro transcribed CAT RNA were combinedwith 1 μg of total RNA from HeLa cells, 1 pmole of oligo GSP1, and DEPCtreated water in a final volume of 14 μl. Mixtures were heated at 70° C.for 10 min to denature secondary structure, and then chilled on ice.Following a brief centrifugation, the remainder of the first strandsynthesis components was added. Reactions were equilibrated to 42° C.for 2 min prior to the addition of SUPERSCRIPT RT. First strandsyntheses were performed in final volumes of 20 μl consisting of 20 mMTris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA, 10 mM DTT, 1pmole GSP1, RNA and 200 units SUPERSCRIPT RT and were incubated 30 minat 42° C. Following first strand conversion, reactions were equilibratedto 55° C. and 2 units E. coli RNase H was added to destroy CAT RNAtemplate. Specific cDNA products were purified, tailed with dCTP, andamplified by PCR as described below.

Primer and unincorporated dNTPs were separated from cDNA using a 30Klow-binding Ultrafee-MC filter unit from Millipore essentially accordingto the manufacturer's recommendations. Four successive wash steps with350 μl 0.1×TE buffer were used to insure sufficient removal of firststand primer. Centrifugations were at 2,000×g for 5 min.

Purified cDNA was recovered with a final rinse of 50 μl sterile H₂O,transferred to a 0.5 ml micro-tube, lyophilized till dry using a SavantSpeed-Vac, then dissolved in 19 μl 10 mM Tris-HCl (pH 8.4), 25 mM KCl,1.25 mM MgCl₂, 50 μg/ml BSA, and 200 μM dCTP. The mixture was denatured2 min, 94° C., then chilled on ice, and the contents collected by briefcentrifugation. Homopolymeric tailing was initiated by addition of 10units TdT and incubated 5 min, 37° C., then 10 min, 65° C.

Tailed cDNA was amplified directly from TdT reactions without priordilution. Following a brief centrifugation to collect tailed cDNA,one-tenth of each tailing reaction, 2 μl aliquots, from were amplifiedby PCR. Amplification reactions were performed in 50 μl volumes composedof 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA], 0.2mM each dNTP, 4 pmoles GSP2, 4 pmoles Anchor Primer (either theGI-Anchor Primer or the G-Anchor Primer), tailed cDNA and 1 unit Taq DNApolymerase (Perkin Elmer-Cetus) Reactions were assembled on ice,overlaid with 50 μl light mineral oil (Sigma), and placed into a DNAThermalcycler (Perkin Elmer Cetus) which had been equilibrated to 94° C.Following an initial 5 min denaturation at 94° C., PCRs were temperaturecycled through 30 cycles as follows: 45 s at 94° C. (denaturation); 25 sat 55° C. (annealing); and 3 min at 72° C. (extension). After the finalcycle an additional 7 min. extension at 72° C. was performed and thenreactions were held at 4° C.

Control reactions omitted either RNA analyte, reverse transcriptase, orTdT. A quantified dc-tailed cDNA target, derived from the CAT RNAanalyte, was used as a positive control.

Analysis of Amplification Products. Following amplification one-fifth,10 μl, of each PCR was analyzed by electrophoresis on a 1.5% agarose/TBEgel. Amplified DNA was visualized by ethidium bromide staining andphotographed.

Results

Based on sequence analysis of the CAT RNA analyte, primers used foramplification were predicted to produce a 930 bp product from dC-tailedCAT cDNA target. This product was observed in PCRs primed with GSP2 andeither the GI-anchor primer or the G-anchor primer. However, sensitivityand band intensity was approximately 10 fold greater in PCRs containingequivalent amounts of target which were amplified using the GI-anchorprimer as compared to amplifications which used the G-anchor primer. The930 bp product was clearly visible in 5′-RACE reactions initiated withas few as 10⁵ copies of RNA analyte when the GI-anchor primer was usedfor amplification. However, product was not visible in a parallelreaction amplified with the G-anchor primer. Band intensity of the 930bp product resulting from 5′-RACE reactions containing 10⁶ copies of CATRNA analyte was approximately 10 fold greater in PCRs performed with theGI-anchor primer as compared to PCRs primed with the G-anchor primer.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A kit for amplifying a nucleic acid moleculecomprising a first primer and a second primer, wherein said first primerhybridizes to a first strand of a nucleic acid template and said secondprimer hybridizes to a second strand of said template, and wherein saidprimers exhibit equalized T_(m)s with respect to each other whenhybridized to said template.
 2. The kit of claim 1, which furthercomprises a thermostable DNA polymerase.
 3. The kit of claim 2, whereinsaid thermostable DNA polymerase is Taq polymerase.
 4. The kit of claim1, wherein said first and/or second primer comprises at least onenucleotide selected from the group consisting of an inosine, ahypoxanthine, a xanthine, and a methylated derivative of a naturallyoccurring base.
 5. A composition for amplifying a nucleic acid templatecomprising a first primer and a second primer, wherein said first primerhybridizes to a first strand of a nucleic acid template and said secondprimer hybridizes to a second strand of said template, and wherein paidprimers exhibit equalized T_(m)s with respect to each other whenhybridized to said template; and a nucleic acid template.
 6. Thecomposition of claim 5, wherein said first and/or second primercomprises at least one nucleotide selected from the group consisting ofan inosine, a hypoxanthine, a xanthine, and a methylated derivative of anaturally occurring base.
 7. The composition of claim 5, wherein saidnucleic acid template is an RNA molecule.
 8. The composition of claim 5,wherein said nucleic acid template is a single-stranded RNA molecule. 9.The composition of claim 5, wherein said nucleic acid template is adouble-stranded RNA molecule.
 10. The composition of claim 5, whereinsaid nucleic acid template is a DNA molecule.
 11. The composition ofclaim 5, wherein said nucleic acid template is a single-stranded DNAmolecule.
 12. The composition of claim 5, wherein said nucleic acidtemplate is a double-stranded DNA molecule.
 13. The composition of claim5, which further comprises a polymerase.
 14. The composition of claim 5,wherein said nucleic acid template is polyadenylated at its 3′ end andwherein one primer comprises a poly-T sequence.
 15. The composition ofclaim 5, wherein said nucleic acid template has been extended tocomprise a 3′ sequence and wherein one primer is capable of hybridizingto said 3′ sequence.
 16. The composition of claim 13, wherein saidpolymerase is a thermostable DNA polymerase.
 17. The composition ofclaim 13, wherein said polymerase is a thermostable Taq polymerase. 18.A reaction mixture for amplifying a nucleic acid template comprising afirst primer and a second primer, wherein said first primer hybridizesto a first strand of a nucleic acid template and said second primerhybridizes to a second strand of said template, and wherein said primersexhibit equalized T_(m)s with respect to each other when hybridized tosaid template; and a nucleic acid template.
 19. The reaction mixture ofclaim 18, wherein said first and/or second primer comprises at least onenucleotide selected from the group consisting of an inosine, ahypoxanthine, a xanthine, and a methylated derivative of a naturallyoccurring base.
 20. The reaction mixture of claim 18, wherein saidnucleic acid template is an RNA molecule.
 21. The reaction mixture ofclaim 18, wherein said nucleic acid template is a single-stranded RNAmolecule.
 22. The reaction mixture of claim 18, wherein said nucleicacid template is a double-stranded RNA molecule.
 23. The reactionmixture of claim 18, wherein said nucleic acid template is a DNAmolecule.
 24. The reaction mixture of claim 18, wherein said nucleicacid template is a single-stranded DNA molecule.
 25. The reactionmixture of claim 18, wherein said nucleic acid template is adouble-stranded DNA molecule.
 26. The reaction mixture of claim 18,which further comprises a polymerase, deoxynucleoside triphosphates, andmagnesium (Mg⁺⁺).
 27. The reaction mixture of claim 18, wherein saidnucleic acid template being amplified is polyadenylated at its 3′ endand wherein one primer comprises a poly-T sequence.
 28. The reactionmixture of claim 18, wherein said nucleic acid template has beenextended to comprise a 3′ sequence and wherein one primer is capable ofhybridizing to said 3′ sequence.
 29. The reaction mixture of claim 26,wherein said polymerase is a thermostable DNA polymerase.
 30. Thereaction mixture of claim 26, wherein said polymerase is a thermostableTaq polymerase.
 31. The kit of claim 1, wherein one or both primerscomprise only nucleotides with altered base pairing characteristics. 32.The kit of claim 31, wherein said nucleotides with altered base pairingcharacteristics are selected from the group consisting of inosine,hypoxanthine, xanthine, 7-methylguanine, and methylated derivatives ofnaturally occurring bases.
 33. The kit of claim 1, wherein one or bothprimers comprise selected from the group consisting of poly-A, poly-C,poly-T, poly-G, and poly-GI.
 34. The composition of claim 5, wherein oneor both primers comprise only nucleotides with altered base pairingcharacteristics.
 35. The composition of claim 34, wherein saidnucleotides with altered base pairing characteristics are selected fromthe group consisting of inosine, hypoxanthine, xanthine,7-methylguanine, and methylated derivatives of naturally occurringbases.
 36. The composition of claim 5, wherein one or both primerscomprise nucleotides selected from the group consisting of poly-A,poly-C, poly-T, poly-G, and poly-GI.
 37. The reaction mixture of claim18, wherein one or both primers comprise only nucleotides with alteredbase pairing characteristics.
 38. The reaction mixture of claim 37,wherein said nucleotides with altered base pairing characteristics areselected from the group consisting of inosine, hypoxanthine, xanthine,7-methylguanine, and methylated derivatives of naturally occurringbases.
 39. The reaction mixture of claim 18, wherein one or both primerscomprise nucleotides selected from the group consisting of poly-A,poly-C, poly-T, poly-G, and poly-GI.